The long-term goal of this investigation is to understand the molecular and enzymatic mechanisms of mutagenesis during the replication of lesion-containing DNA. DNA damage has two roles in mutagenesis, causing misincorporation during DNA replication or repair synthesis on non-instructive DNA templates and inducing cellular functions (SOS functions) that cause normally error-free DNA replication and repair to become error-prone. Understanding mechanisms of mutagenesis will require knowing not only the identities and functions of induced proteins but also how these proteins in SOS-induced cells interact with the DNA replication apparatus and lesions in template DNA. Single-stranded DNA phages containing cloned inserts of E. coli chromosomal and plasmid genes will be used as forward and reverse mutation systems to study targeted mutagenesis by ultraviolet damage in phage DNA and untargeted mutagenesis of phage DNA in SOS-induced (UV-irradiated) cells. Mutagenesis of nonessential cloned genes in phage DNA can be detected in vivo, by infection with hybrid phage, or by transfection with hybrid phage DNA in either single- or double-stranded form. Hence, the systems will provide sensitive assays to compare mutation sites and damage sites in phage DNAs, to determine the host genetic requirements for targeted and untargeted mutagenesis, and to study mutation reactions during in vitro DNA replication. The proposed investigation is a basis for studying the biochemical reactions that give rise to mutations in DNA damaged by mutagens and carcinogens. That low levels of such agents may induce SOS-like phenomena in mammalian cells is a health concern that can only be assessed when their mechanisms of action are fully understood.